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Visualization of Nascent Transcripts on Drosophila Polytene Chromosomes Using BrUTP Incorporation BioTechniques 29:934-936 (November 2000)
Polytene chromosomes from the salivary gland cells of Drosophila third instar larvae have been used extensively for cytological and genetic studies. Polytene chromosomes have also been used to study transcription because an actively transcribing gene is often manifested as a localized region of chromatin decondensation or “puff”. Several methods have been developed to detect sites of active transcription on polytenes, including direct autoradiography following tritiated nucleotide incorporation (7), hybridization of tritiated RNAs (6) and detection of RNA/DNA hybrids by immunofluorescence microscopy (8). Each of these methods has one or more drawbacks. Direct autoradiography is a fairly difficult technique and requires exposure times of up to several weeks. It is often difficult to precisely map the sites of transcription because of the migration of the decay emission. In addition, it is difficult to detect weakly transcribing sites because of high background. Analyzing RNA synthesis by in situ hy934 BioTechniques
bridization of labeled RNAs can be compromised if certain RNAs are lost during the RNA isolation procedure or if nonspecific hybridization or binding of the probe occurs (2). The detection of actively transcribing sites using antibodies that detect RNA/DNA hybrids has produced good results but is limited in that it is not suited for pulse-chase experiments and that commercial antibodies recognizing the hybrids are not readily available. Many of the disadvantages of studying nascent transcripts have been overcome through the use of nucleotide analogue incorporation followed by immunofluorescence detection. A comparison of biotin-11-UTP, digoxigenin11-UTP and bromo-UTP (BrUTP) indicated that of these three modified nucleotides, BrUTP was incorporated into transcripts the most efficiently in mammalian cells (5). While early studies using mammalian cells used permeabilization (5) or direct microinjection (9) to get BrUTP into cells, more recent methods have used the cationic lipid DOTAP (4) or the non-liposomal agent FuGENEÔ 6 (Roche Molecular Biochemicals, Laval, PQ, Canada) (3) to load BrUTP. All of these studies led us to investigate whether BrUTP could be used to label nascent transcripts on Drosophila polytene chromosomes. We have adapted the method described by Haukenes and co-workers (4) to label transcripts on polytene chromosomes. Salivary glands from wandering Drosophila melanogaster third instar larvae were first dissected out in a drop of modified TB1 buffer (15 mM HEPES, pH 6.8, 80 mM KCl, 16 mM NaCl, 5 mM MgCl2 and 1% PEG 6000) (1). All chemicals used in this procedure were from Sigma (St. Louis, MO, USA) unless otherwise noted. After dissection, isolated glands were allowed to sit for at least 1 h at room temperature in a depression slide containing 200 mL TB1 before labeling with BrUTP. The depression slide was kept in a humidified chamber to prevent evaporation of the buffer. In the transcription inhibition experiments, cells were incubated in TB1 for 1 h, then TB1 plus inhibitor [either actinomycin D (10 mg/mL) or a-amanitin (50 mg/mL)] for 1 h before labeling for 20 min in the presence of inhibitor.
To make our standard 10 mM BrUTP labeling solution (50 mL), the following were combined in a 1.5-mL microcentrifuge tube: 35 mL TB1, 10 mL DOTAP (Roche Molecular Biochemicals) and 5 mL 100 mM BrUTP. The components were pipetted up and down gently to mix, and the mixture was incubated for 10 min at room temperature to allow the DOTAP to complex with the BrUTP. Early experiments also involved testing a lower concentration (1 mM) of BrUTP in the labeling mixture and mixtures containing different concentrations of BrUTP with and without DOTAP. In a typical experiment, one or two pairs of salivary glands were transferred to a 50-mL BrUTP labeling mixture in a depression slide using a siliconized pipet tip or forceps. After a specified labeling time at room temperature (typically 15 or 20 min), glands were transferred to a drop of fixative (50% acetic acid/3.7% formaldehyde) on a siliconized (SigmacoteÒ; Sigma) coverslip for 1–2 min and then squashed on a microscope slide. When “partial” squashes were performed (i.e., no pressure on the coverslip was applied to keep the cells intact), the fixation time was 2–3 min. Preparations were frozen in liquid nitrogen to facilitate the removal of the coverslip and then stored in 95% ethanol until immunostaining was performed. For the RNase-treated specimens, slides containing fixed squashed salivary gland cells that had incorporated Bromo-Uridine were washed briefly with 2´ SSC. Next, the slides were incubated for 2 h with 125 mL 1 mg/mL DNase-free RNase A (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in 2´ SSC. The slides were then rinsed with 2´ SSC and returned to a Coplin jar with 95% ethanol. Chromosome squash preparations were immunostained essentially following the method described by Westwood and co-workers (11). Squashes were first incubated with an anti-bromodeoxyuridine (anti-BrdU) mouse monoclonal antibody (Roche Molecular Biochemicals) diluted to a final concentration of 2 mg/mL in BTP (0.5% BSA, 0.1% Tween Ò 20 in PBS) for 1–2 h at room temperature. We have subsequently used anti-BrdU antibodies from Chemicon International (Temecula, CA, Vol. 29, No. 5 (2000)
USA) and the Developmental Studies Hybridoma Bank, Department of Biology, University of Iowa (antibody no. G3G4) and have obtained equally good results. The secondary antibody used was a fluoroscein isothiocyanate (FITC)-conjugated F(ab¢)2 goat antimouse IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted 1:200 in BTP. After immunostaining for Bromo-Uridine, the DNA was stained for 5 min with 1 mg/mL Hoechst 33342 in PBS and then mounted in 0.1% phenylenediamine in 70% glycerol/30% PBS. Images were recorded on FujichromeÒ Sensia 400 ISO slide film using a NikonÒ Microphot fluorescence microscope and a
Nikon Plan 40´ objective. Exposure times ranged from 6 to 8 s for Hoechst staining (with neutral density filters) and from 22 to 30 s for FITC staining. The 35-mm slides were digitized using a Nikon LS30 scanner and, when necessary, adjusted for brightness and contrast using AdobeÒ Photoshop Ò. Figure 1 shows the results from a variety of different BrUTP labeling experiments. Previous studies using mammalian cells (4,5,9) have shown that anti-BrdU antibodies can also recognize Bromo-Uridine that has been incorporated into RNA. In Figure 1, panels A–H, fixed salivary glands were placed between a coverslip and a slide but were not squashed to maintain the
Figure 1. Bromo-Uridine-labeled nascent transcripts in the nuclei of Drosophila salivary gland cells and on polytene chromosomes. Salivary glands were dissected fromD. melanogaster third instar larvae and incubated in organ culture (with or without chemical treatments) for 1 h before BrUTP labeling. Glands were labeled in a 10-mM BrUTP/DOTAP mixture for 20 min before fixation and immunostaining. Panels A–H are intact salivary glands cells, while panels I and J are squashed preparations showing chromosome spreads. Bromo-Uridine (BrU) incorporation is shown in panels A, C, E, G, I and J and was detected using an anti-BrdU antibody. The cells in panels A, C, E and G were also stained with Hoechst 33342 to visualize DNA as shown in panels B, D, F and H, respectively. Panels C and D show cells treated with actinomycin D (10 mg/mL), and panels E and F show cells treated with a-amanitin (50 mg/mL) before and during BrUTP labeling. Panels G and H show cells treated with 1 mg/mL DNase-free RNase A after they had incorporated Bromo-Uridine. Panel J is a chromosome spread from glands treated with 1 mM 20-hydroxyecdysone for 1 h before and during BrUTP labeling. The ecdysone-inducible puffs at 74EF and 75B are marked. Bar = 10 mM. Vol. 29, No. 5 (2000)
integrity of the nucleus and the rest of the cell. In Figure 1, panel A, one can clearly make out bright anti-BromoUridine staining in the nuclei of two adjacent salivary gland cells. The antiBromo-Uridine staining appears to be predominantly localized on the chromatin because the staining pattern follows the pattern of DNA staining (Figure 1, panel B). In a typical experiment, Bromo-Uridine incorporation was observed in 20%–50% of the cells. Using a tenfold lower concentration of BrUTP in the labeling solution resulted in about the same fraction of cells incorporating Bromo-Uridine, but the staining was less intense (results not shown). Using Bromo-Uridine itself rather than BrUTP in the labeling mixture (with or without DOTAP) resulted in a lower percentage of cells showing incorporation and less intense staining in those cells that did incorporate the BromoUridine (results not shown). Leaving out BrUTP (or Bromo-Uridine) from the labeling mixture resulted in no staining (not shown). To confirm that the observed staining pattern did represent nascent RNAs, experiments using inhibitors of transcription were performed. The addition of actinomycin D, a drug that inhibits transcription by all three RNA polymerases, resulted in a low level of fluorescence throughout the nucleus and cytoplasm with no specific staining pattern in the nucleus (Figure 1, panel C). The addition of aamanitin, which at the concentration used in this experiment should inhibit virtually all transcription by RNA polymerase II but not polymerase I (10), resulted in a small amount of anti-BromoUridine staining within the nucleus (Figure 1, panel E). Figure 1, panel E, and a number of other preparations including full squashes (not shown), indicate that this staining is likely localized to specific regions within the nucleolus. To further confirm that the anti-BrdU fluorescent staining patterns represent nascent transcripts, salivary gland cells that had been labeled with BrUTP were treated with DNase-free RNase before immunostaining with anti-BrdU antibodies. No Bromo-Uridine staining was detected in these preparations (Figure 1, panel G). Thus, the results of the RNA polymerase inhibitor experiments and the RNase treatment are consistent with BioTechniques 935
Benchmarks the interpretation that the anti-BrdU staining seen corresponds to BromoUridine that has been incorporated into nascent transcripts and not incorporation of BrUTP into DNA or nonspecific binding within the cells. Figure 1, panel I, shows the BromoUridine staining pattern on polytene chromosomes in a salivary gland cell that has been squashed to spread the chromosomes. Approximately 250 distinct bands or sites of Bromo-Uridine incorporation can be made out along with other staining that is less distinct. There is a wide range of staining intensities along the chromosomes, most likely indicating that transcription is occurring at different rates at the various sites and/or that there are multiple genes being transcribed at a particular site. In Figure 1, panel J, salivary glands were incubated in TB1 containing 1 mM 20-hydroxyecdysone (ecdysone) for 1 h before labeling for 20 min with BrUTP (in the presence of ecdysone). Ecdysone is known to induce six “early” developmental puffs. The two largest puffs occur at 74EF (a site that encodes an ets-related transcription factor) and 75B (a site that encodes a steroid receptor DNA-binding protein). These puffs are easily identifiable and show a tremendous amount of Bromo-Uridine incorporation. The results presented here clearly demonstrate that nascent transcripts can be visualized on polytene chromosomes of Drosophila through Bromo-Uridine incorporation. The level of resolution of transcript localization is unsurpassed compared to other methods, and in many instances, the fluorescent band observed on the chromosomes can be attributed to the transcription of a single gene. Using antibodies to detect RNA/ DNA hybrids offers similar resolution, but the BrUTP labeling method has the added advantage of being able to visualize nascent transcripts, thus making it possible to compare global transcription patterns before and after a specific gene-inducing event. However, it does have the disadvantage of having to add BrUTP to perform the experiment. The BrUTP labeling method may also be suitable for pulse-chase experiments. In addition, the reagents used to perform these experiments are readily available through commercial sources. 936 BioTechniques
REFERENCES 1.Bonner, J.J. 1981. Induction of Drosophila heat-shock puffs in isolated polytene nuclei. Dev. Biol. 86:409-418. 2.Bonner, J.J. and M.L. Pardue. 1977. Polytene chromosome puffing and in situ hybridization measure different aspects of RNA metabolism. Cell 12:227-234. 3.Haukenes, G. and K.-H. Kalland. 1998. Labelling of eukaryotic transcripts with BrUTP using a non-liposomal transfection reagent. Biochemica 3:45-46. 4.Haukenes, G., A.M. Szilvay, K.A. Brokstad, A. Kanestrom and K.H. Kalland. 1997. Labeling of RNA transcripts of eukaryotic cells in culture with BrUTP using a liposome transfection reagent (DOTAP). BioTechniques 22:308-312. 5.Jackson, D.A., A.B. Hassan, R.J. Errington and P.R. Cook. 1993. Visualization of focal sites of transcription within human nuclei. EMBO J. 12:1059-1065. 6.Pardue, M.L., S.A. Gerbi, R.A. Eckardt and J.G. Gall. 1970. Cytological localization of DNA complementary to ribisomal RNA in polytene chromosomes of diptera. Chromosoma 29:268-290. 7.Pelling, C. 1959. Chromosomal synthesis of ribonucleic acid as shown by incorpration of uridine labeled with tritium. Nature 184:655656. 8.Rudkin, G.T. and B.D. Stollar. 1977. High resolution detection of DNA-RNA hybrids in situ by indirect immunofluorescence. Nature 265:472-473. 9.Wansink, D.G., W. Schul, I. van der Kraan, B. van Steensel, R. van Driel and L. de Jong. 1993. Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. J. Cell Biol. 122:283-293. 10.Weaver, R.F. 1999. Molecular Biology, p. 268-272. WCB McGraw-Hill, New York. 11.Westwood, J.T., J. Clos and C. Wu. 1991. Stress-induced oligomerization and chromosomal relocalization of heat- shock factor. Nature 353:822-827.
This work was supported by a grant from NSERC Canada to J.T.W. Address correspondence to Dr. J. Timothy Westwood, Department of Zoology, University of Toronto at Mississauga, 3359 Mississauga Rd., Mississauga, ON, Canada L5L 1C6. email:
[email protected] Received 4 February 2000; accepted 2 August 2000.
W.Y. Chang, N.A. Winegarden, J.P. Paraiso, M.L. Stevens and J.T. Westwood University of Toronto Mississauga, ON, Canada
Microarray-Based Detection of Select Cardiovascular Disease Markers BioTechniques 29:936-944 (November 2000)
Assessment of the genetic risk for cardiovascular disease is complex. Genes linked to hypertension and/or thrombophilia are among the key components. Both the angiotensinogen (8) and a-adducin (3) genes play a role in hypertension. An adenine to guanine mutation in the promoter region at nucleotide-6 of the angiotensinogen gene (AGT-6) alters the binding of the nuclear protein, which results in increased gene transcription and elevated plasma angiotensinogen levels (7). a-Adducin (ADD) regulates sodium-potassium transport in the renal tubule through modifications in the actin cytoskeleton; individuals with a point mutation (G460T) in the ADD gene are reported to be more sensitive to changes in sodium balance (4). Proteolytic inactivation of factor V by activated protein C is a critical regulatory step that limits clot formation during activation of the coagulation cascade. A point mutation (Leiden) in the factor V gene (G1,691A) predicts the generation of an abnormal factor V molecule in which arginine at amino acid position 506 is replaced by glutamine. This mutation (FVL) renders factor V resistant to proteolytic inactivation by activated protein C and shifts the coagulation cascade toward one of clot formation (i.e., the clinical condition of thrombophilia) (2). DNA microarray technology provides an ideal platform for investigating the combination of polymorphisms and mutations described above. We recently reported a strategy based on the combined use of multiplex PCR/ligase detection reaction (PCR/PCR/LDR) with “zip-code” hybridization to DNA probes on glass slides for the simultaneous detection of such genetic variants (5,6). This approach provides for an accurate, inexpensive and high-throughput assay that does not exhibit false-positive or falsenegative signals, thus making it highly suitable for gene-based testing in highincidence, low-complexity diseases. Vol. 29, No. 5 (2000)
